Lignocellulose is the most abundant biopolymer on earth. Lignocellulose is the major structural component of woody plants and non-woody plants such as grass. Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose, and lignin. Large amounts of lignocellulosic residues are produced through forestry, timber and pulp and paper industries and agricultural practices (straw, stover, bagasse, chaff) and many agroindustries. Also municipal waste contains fractions that can be considered as lignocellulose residues, such as paper or cardboard waste, garden waste or waste wood from construction. Due to high abundance and low price lignocellulosic residues are preferred materials for production of biofuels. In addition, dedicated woody or herbaceous energy crops with biomass productivity have gained interest as biofuel use.
The production of biofuels, especially ethanol, from lignocellulosic materials by microbial fermentations has been studied extensively. The greatest challenge for utilization of lignocellulosics for microbiological production of biofuels or biofuel feedstocks lays in the complexity of the lignocellulose material and in its resistance to biodegradation. In lignocellulose, cellulose (20-50% of plant dry weight) fibers are embedded in covalently found matrix of hemicellulose (20-40%), pectin (2-20%) and lignin (10-20%) forming very resistant structure for biodegradation. Further, the sugar residues of hemicellulose contain a varying mixture of hexoses (e.g., glucose, mannose and galactose), and pentoses (e.g., arabinose and xylose) depending on the biomass.
The pre-treatment of lignocellulosic material with high yield to sugars that are utilizable by micro-organisms represents one of the highest challenges. Significant cost reductions are needed in the costs of enzymes needed in hydrolysis of sugar polymers to sugar monomers that are utilizable by desired microorganisms. Further, the economically feasible production of biofuels from lignocellulosic materials requires efficient conversion of all the main carbohydrate constituents of this complex material to biofuels.
Enzymatic hydrolysis of the lignocellulosic material is typically performed in a separate step from biofuel production process by commercial enzymes bought and produced outside the actual biofuel production process.
Certain microorganisms can produce lipids from organic molecules, such as sugars derived from lignocellulose. Certain microorganisms, typically yeast, fungi or bacteria, can efficiently convert both C6 and C5 sugars in lignocellulosic materials to oil. Oil produced by heterotrophic microorganisms is often called as single cell oil or microbial oil. Single cell oil production process using heterotrophic microorganisms comprises cultivating microorganisms in aerated bioreactors, allowing cells to accumulate lipids, harvesting lipid-rich cells and recovering oil from cells. Microorganism-based lipids (i.e. single cell oils) can be used as raw materials for production of biofuels such as biodiesel, renewable diesel or bio jet fuel.
Lignocellulose hydrolysates have been utilized also in the production of single cell oils. Lignocellulose hydrolysis has been typically carried out by pre-treating the lignocellulosic material to monomeric sugars prior feeding to bioprocess.
Patent publication US2009217569 describes single cell oil production from various lignocellulosic and other material hydrolysates, such as straw, wood, pulp and paper industry residues, recycled fibres, municipal waste, algae biomass. Manufacturing biofuel comprises treating source material with water, acid or alkali and contacting filtrate or precipitate with lipid-producing microorganism. Patent publication US2009064567 describes single cell oil production from cellulose material hydrolysates for biodiesel and jet biofuel production by Stramenopiles. US20090011480 describes single cell oil production by heterotrophically grown algae and fungi from depolymerised lignocellulosic materials, such as straw, wood, pulp mill waste, switchgrass. CN101148630 describes single cell oil production from wheat, corn or rice straw hemicellulose hydrolysates, obtained by steam explosion, by bacteria or fungi.
Further, in the prior art has been described lipid production directly from polymeric sugars in lignocellulose, such as xylan by Fall et al. (1984), or cellulose by Lin et al. (2010).
WO2010042842 describes production of single cell oil from lignocellulose hydrolysates by mixed culture of microorganism(s) capable of degrading polymeric sugars in lignocellulose and at least one algae species. The culture is grown in successive aerobic and anaerobic cultivations, where fatty acids are produced from sugars and from anaerobic fermentation products.
WO2010006228 describes sequential production of biofuels from lignocelluloses. In first stage, anaerobic fermentation with organisms capable of producing alcohols from polymeric sugars in lignocellulose hydrolysates, in second stage, the spent culture medium, possibly containing at least one fermentation product, is treated with algae in order to accumulate single-cell oils.
The presence of contaminating non-lipid producing microbes in fermentation broth may influence the oil productivity and yield since the non-lipid producing microbe compete with oil producing microorganisms (oleaginous microbes) on sugars in the lignocellulose hydrolysates and thus making the process less feasible.
There is therefore a need for method of controlling the culture of micro-organisms in the single cell oil production process in such a way that the proliferation of the oleaginous microbes is favoured over the proliferation of non-oleaginous microbes